Use of mutated antithrombins for treating or preventing coagulation disorders

ABSTRACT

Use of a mutated antithrombin having substantially no activity, in particular no anticoagulant activity, possibly in association with an anticoagulant, for the preparation of a drug intended for the prevention or treatment of pathologies linked to or associated with coagulation disorders.

The present invention relates to the use of mutated antithrombins for treating or preventing coagulation disorders.

Antithrombin plays an essential role in maintaining the fluidity of blood. Blood coagulation is mediated by a series of serine proteases. Antithrombin is a potent inhibitor of Factors VIIa IXa, Xa, XIa, XIIa and IIa (thrombin).

The physiological importance of antithrombin in preventing excessive coagulation is revealed by studies of individuals whose antithrombin levels are decreased due to heredity or acquired deficiency. Such persons are prone spontaneous thrombosis and the associated risks of disseminated intravascular coagulation, cardiac infarction, cerebrovascular accident and pulmonary embolism.

It was well known that the therapeutic administration of anticoagulants (heparins, pentasaccharide Fondaparinux Arixtra® and pentasaccharide derivatives) prevents the formation of clots and the extension of existing clots within the blood and thus, is useful in the clinical prevention and management of venous thromboembolic disease.

The anticoagulant effect of antithrombin is enhanced by heparins and derivatives, in particular pentasaccharide, which greatly increases the rate of inhibitor-protease complex formation.

Only a fraction of heparin molecules are functional in this regard due to the presence in their structure of a specific pentasaccharide moiety capable of binding antithrombin with high affinity and inducing active conformational changes in the antithrombin.

The crystal structure of the free antithrombin and of antithrombin complexed with the above-mentioned pentasaccharide reveals that the pentasaccharide binding to an allosteric site on the inhibitor transmits conformational changes to a reactive proteinase binding loop on the inhibitor surface that enhances the loop accessibility to proteinases (Skinner, R., Abrahams, J-P., Whisstock, J. C., Lesk, A. M., Carrell, R. W., and Wardell, M. R. (1997) J. Mol. Biol. 266 601-609; Jin, L., Abrahams, J. P., Skinner, R., Petitou, M., Pike, R. N., and Carrell, R. W., (1997) Proc. Natl. Acad. Sci., U.S.A. 94, 14683-14688).

Heparin or the pentasaccharide is used as an anticoagulant in several clinical applications such as for treatment of various thrombotic diseases, unstable angina, and thrombosis prophylaxy in medical/surgical patients, for thrombosis management related to extracorporeal circulatory assistance or dialysis devices, for treatment of myocardial infarction (primarily and adjunctively with various thrombolytic agents).

However, the use of heparin can lead to undesired complications and in particular to hemorrhages. In some cases, this is due to the fact that the dosage of administered heparin is not appropriate, which might result in a limitation of the successful clinical use of heparin.

The only antidote available for heparin neutralisation is protamin. However, it is known that the protamin cannot neutralize the pentasaccharide (Giangrande PL. Fondaparinux (Arixtra): a new anticoagulant. Int J Clin Pract 2002; 56: 615-617), and is associated with various side effects (hemodynamic instability, anaphylactic shock and bleeding risk linked to an anticoagulant activity of protamin in case of overdosing), and in particular the administration of important doses of protamin increases the hemorrhage.

At this day, no completely suitable antidotes to heparin and to the pentasaccharide of heparin have been found and more particularly no suitable antidotes effective in vivo have been described, knowing that in the field of coagulation, in vitro tests are never sufficient to predict the in vivo effects.

For example, in the document (Krupinski et al. Antithrombotic effects of three thrombin inhibitors in a rat model of laser-induced thrombosis” Haemostasis 1989; 19 (2): 74-82) it is demonstrated that the anticoagulant effect of hirudin, NAPAP and argidipine do not differ in vitro, whereas ex vivo, the anticoagulant effect of hirudin is most important.

Another document (Yamashita et al. “The antithrombotic effect of potent bifunctional thrombin inhibitors based on hirudin sequence, P551 and P532, on He—Ne Laser induced thrombosis in rat mesenteric microvessels” Thrombosis Research 90 (1998) 199-206) suggests that “the interactions between thrombin and the inhibitors in vivo are different from those in vitro”.

One of the aims of the inventions is to provide safe and suitable antidotes to heparin and to the pentasaccharide.

One of the aims of the inventions is to provide suitable antidotes to heparin and to the pentasaccharide, able to neutralize heparin and pentasaccharide, easy to use and devoid of side effects.

This is achieved through the use of appropriate mutated antithrombins.

More precisely, the present invention relates to the use of a mutated antithrombin having substantially no activity, in particular no anticoagulant activity, possibly in association with an anticoagulant, for the preparation of a drug intended for the prevention or treatment of pathologies linked to or associated with coagulation disorders.

The term <<mutated antithrombin>> designates a human antithrombin comprising at least a substitution, insertion and/or deletion of one or more amino acids within its amino acid sequence.

The said mutated antithrombins can be prepared according to the method described in the experimental part I/.

The human antithrombin sequence is described in Olds R. J., Lane D. A., Chowdhury V., De Stefano V., Leone G. and Thein S. L. “Complete nucleotide sequence of the antithrombin gene: evidence for homologous recombination causing thrombophilia>> Biochemistry. 32 (16), 4216-4224 (1993).

The human antithrombin sequence of the invention is an Homo sapiens serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1), mRNA. Accession NM 000488, Version NM 000488.2, GI:50541941.

There are many references which describe said DNA sequence (with signal peptide) but they are not absolutely identical because of the many natural polymorphisms of antithrombin which generally do not change the properties of the antithrombin.

The human aminosequence of antithrombin presents two forms: a “short form” (SEQ ID NO: 2) which does not comprise a signal peptide and a “long form” (SEQ ID NO: 26) which includes a signal peptide.

The signal peptide comprises 32 amino acids and is necessary for antithrombin secretion. It is removed during antithrombin processing and the plasma antithrombin circulates as the <<short form>>.

Accordingly, in the present invention, the mutated antithrombins amino acid sequences, represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24, do not comprise the signal peptide and the mutated antithrombins amino acid sequences, represented, by SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 et 48, include the signal peptide.

The mutated antithrombin of the invention can be used in association or not with an anticoagulant.

The term <<mutated antithrombin>> as used herein, designates:

mutated antithrombins which are different from mutants of antithrombin known in the art in that said mutated antithrombins of the invention have no anticoagulant activity, and able to compete in vivo with plasma antithrombin for glycosaminoglycans binding, and furthermore,

when mutated antithrombins of the invention are used with anticoagulant, mutated antithrombins have, in addition to the above mentioned properties, the property to compete in vivo with plasma antithrombin with respect to the binding to the anticoagulant.

It has unexpectedly been found that said mutated antithrombins of the invention are able to compete in vivo with plasma antithrombin and can be used to prevent or treat excessive fluidity of blood.

Moreover, it has unexpectedly been found that the said mutated antithrombins are able to compete with plasma antithrombin with respect to the binding to the anticoagulant and that the therapeutic administration of mutated antithrombins, in association with anticoagulants, prevents the undesired complications, and in particular hemorrhages, resulting from side effects of said anticoagulant.

The expression <<mutated antithrombin having substantially no activity>> designates a mutated antithrombin which has lost its capacity to inhibit coagulation.

An example of a test to determine the absence of anticoagulant activity is described in the experimental part II/a) of the present application.

The expression <<anticoagulant>> designates a substance that prevents coagulation and can be used in vivo as a medication for thrombotic disorders.

The expression <<pathologies linked to or associated with coagulation disorders>> as used herein designates a coagulation excess or defect, caused by abnormalities in the composition of the blood, the quality of the vessel wall and/or the nature of the blood flow.

More preferably, the present invention relates to the use of a mutated antithrombin at a concentration from about 0.1 to about 10 fold the concentration of the plasmatic antithrombin, particularly from about 1 to about 5 fold the concentration of the plasma antithrombin.

The concentration of the plasma antithrombin is from about 150 μg/ml to about 350 μg/ml, particularly about 300 μg/ml.

In an advantageous embodiment, the present invention relates to the use of a mutated antithrombin, in association with an anticoagulant, for the preparation of a drug intended for the treatment or prevention of hemorrhagic disorders and related pathologies, resulting from side effects of said anticoagulant.

In many clinical situations, heparin and in particular the pentasaccharide of heparin, is effective for prevention and treatment of thromboembolic events, but would require dose adjustment.

If the dosage of administrated heparin is not appropriate, heparin can cause serious adverse effects, in particular hemorrhage.

The use of a mutated antithrombin such as described above, allows to neutralize in particular heparin or the pentasaccharide and to prevent and/or treat hemorrhage.

The expression <<hemorrhagic disorders and related pathologies resulting from side effects of said anticoagulant>> designates bleeding complications. Bleeding side effects ranged in severity from local hematomas to major hemorrhagic events including death.

The present invention relates to the use of the above mentioned mutated antithrombin, wherein said mutated antithrombin has the ability to bind to the anticoagulant and to shift, in particular in vivo, the binding between plasma antithrombin and said anticoagulant.

The expression <<mutated antithrombin has the ability to bind to the anticoagulant>> designates a mutated antithrombin which binding affinity for the anticoagulant is similar or higher than that of plasma antithrombin for the anticoagulant.

A test for the determination of the binding between the mutated antithrombin and the anticoagulant is for instance the measure of the intrinsic fluorescence of the mutated antithrombin in the absence or in the presence of increasing concentration of anticoagulant (Meagher, Beechem, Olson and Gettins J B C 1998, 23283-232).

The term <<to shift in vivo the binding>> corresponds to a competition between plasma antithrombin and mutated antithrombin for the anticoagulant binding.

A test for the determination of the shift of the binding by mutated antithrombins is for instance the measure of the plasma antithrombin anti factor Xa inhibitory activity in a plasma containing the anticoagulant.

This test is described in experimental part (II/b for in vitro experiments and III/2 for in vivo experiments).

It is important to investigate, in vivo in an experimental model, the effect of the mutated antithrombin on plasma antithrombin-anticoagulant complex because in vitro tests are not sufficient to predict the in vivo effect of the mutated antithrombins of the invention.

The term <<plasma antithrombin>> corresponds to an endogenous glycoprotein produced by the liver. The alpha-antithrombin is the dominant form in blood plasma. The physiological target proteases of plasma antithrombin are those of the intrinsic coagulation system, namely the actived forms of factor VII, (VIIa), Xa, IXa, VIIa, XIa, XIIa and the thrombin. Proteases inactivation results as a consequence of the trapping the protease in an equimolar covalent complex with plasma antithrombin in which the active site of the protease enzyme is inaccessible to its usual substrate. It is known that plasma antithrombin has a high affinity for heparin and that inhibition rates on thrombin and factor Xa can be accelerated up to 10 000 fold in the presence of heparin.

In an advantageous embodiment, the present invention relates to the use of the above-mentioned mutated antithrombin in association with a mutant of antithrombin, the amino acid sequence of which differs from that of said mutated antithrombin, wherein said mutated antithrombin has the ability to shift, in particular in vivo, the binding between plasma antithrombin and said anticoagulant and the ability to shift, in particular in vivo, the binding between said mutant of antithrombin and said anticoagulant.

The term <<mutant of antithrombin>> designates all the variants of antithrombin with increased anticoagulant activity and which are different from endogenous mutants of antithrombin.

The term <<amino acid sequence of which differs from that of said mutated antithrombin>> designates that there is at least one amino acid which is different, by its nature, or its presence or absence, when comparing the sequence of the mutated antithrombin of the invention and the sequence of the mutant of antithrombin.

The present invention relates to the use of the above-mentioned mutated antithrombin, wherein said mutated antithrombin has substantially lost factor Xa inhibitory activity and thrombin (IIa) inhibitory activity.

In particular, the present invention relates to the use of the above-mentioned mutated antithrombin, wherein said mutated antithrombin has substantially lost its inhibitory activity of serine proteases involved in the blood coagulation.

The expression <<factor Xa inhibitory activity>> designates the ability of antithrombin to interact with factor Xa and to irreversibly trap and inactivate this enzyme.

The expression <<thrombin inhibitory activity>> designates the ability of antithrombin to interact with thrombin and to irreversibly trap and inactivate this enzyme.

A test for the determination of the loss of the thrombin inhibitory activity is for instance an assay performed in vitro, in which thrombin is incubated with an excess of the tested mutated antithrombins in the presence or absence of pentasaccharide and in the presence of a given concentration of chromogenic substrate S2238. When mutated antithrombin exhibits thrombin inhibitory activity, absorbance at 405 nm resulting from S2238 cleavage increases according to a first-order exponential as a function of time. At the opposite, when mutated antithrombin loses its thrombin inhibitory activity or in the absence of inhibitor, absorbance at 405 nm follows a linear increase during the period of the assay.

A test for the determination of the loss of the FXa inhibitory activity is for instance an assay performed in vitro, in which thrombin is incubated with an excess of the tested mutated antithrombin in the presence or absence of pentasaccharide and in the presence of a given concentration of chromogenic substrate S2765. When mutated antithrombin exhibits FXa inhibitory activity, absorbance at 405 nm resulting from S2765 cleavage increases according to a first-order exponential as a function of time. At the opposite, when mutated antithrombin loses its FXa inhibitory activity or in the absence of inhibitor, absorbance at 405 nm follows a linear increase during the period of the assay.

According to an advantageous embodiment, the use of the mutated antithrombin of the present invention is characterized in that the value of the dissociation equilibrium constant (Kd) of the complex resulting from the binding of said mutated antithrombin with said anticoagulant, at a given ionic strength, in particular at physiological ionic strength, is similar or lower than the value of the Kd of the complex between plasma antithrombin and said anticoagulant.

The corresponding dissociation equilibrium constant (Kd) is measured according to the method described by monitoring the change in intrinsic protein fluorescence upon binding of heparin (Meagher, Beechem, Olson and Gettins J B C 1998, 23283-23289).

Heparin (or pentasaccharide) binding to antithrombin is assessed by titrating fixed level of antithrombin with heparin and monitoring the tryptophan fluorescence increase signaling heparin binding. Excitation and emission wavelengths 280 and 340 nm respectively are used and titration curves are fitted to the quadratic equilibrium binding equation to obtain the Kd (Olson S T, Björk I and Shore J D, 1993, Methods enzymol., 222, 525-560).

Similarly, the dissociation equilibrium constant (Kd) of the complex, resulting from the binding of plasma antithrombin with said anticoagulant, is measured using the same method, with purified plasma antithrombin.

The expression <<a given ionic strength, in particular at physiological ionic strength>> designates an ionic strength corresponding to 150 mM NaCl (according to the Vidal dictionary, injectable sodium chloride solution should be at 0.9%, corresponding to 154 mM).

The expression <<is similar or lower than>> designates a Kd which is between 5 fold higher to 500 fold lower, more particularly 2 to 50 fold lower to the Kd determined for plasma antithrombin, in our experimental conditions.

The mutated antithrombin of the invention can be active even if the Kd is 5 fold higher than that of the plasma antithrombin provided that the corresponding doses required to shift the binding between plasma antithrombin and said anticoagulant are therapeutically compatible.

According to an advantageous embodiment, the use of the mutated antithrombin of the present invention is characterized in that the value of the Kd of the complex resulting from the binding of said mutated antithrombin with said anticoagulant, at a given ionic strength, in particular at physiological ionic strength, is similar or lower than the value of the Kd of the complex between said mutant of antithrombin and said anticoagulant.

The Kd value of the complex between mutant of antithrombin and said anticoagulant is estimated according to the same procedure above-mentioned.

According to an advantageous embodiment, said anticoagulant is chosen among heparins, heparins derivatives, in particular unfractioned heparins, low molecular weight heparins, the anticoagulant pentasaccharide (Fondaparinux), (Org31540/SR90107, Arixtra®) and its derivatives (Idraparinux: SANORG 34006), and heparinoids (Danaparoid sodium ORG 10172).

Other examples of anticoagulants which can be used are pentasaccharide with anti-Xa activity, pentasaccharide with anti-Xa activity and anti-IIa activity (Chritian Noti and Peter Seeberger, Chemistry and biology. 2005; 12: 731-756) (Synthetic analogues of the Antitrombin III-Binding Pentassacharide Sequence of heparin” Ronald G. M. van Amsterdam; Gerard M. T. Vorgel; Arie Visser; Wim J. Kop; Marc T. Buiting; Dirk G. Meuleman. Atheroscler Thromb Vasc Biol. 1995; 15: 495-503).

Among pentasaccharide derivatives, there can be mentioned compounds N° 33-51-78-81-84 and 93 described in Noti et al. 2005 and compounds 32543 and 32271 described in van Amsterdam et al. 1995 (see references above).

According to an advantageous embodiment, the pathologies linked to or associated with coagulation disorders are among arterial or venous thrombotic disorders such as pulmonary embolism, deep vein thrombosis, myocardial infraction, unstable angina, stroke, disseminated intravascular coagulation and among hemorrhagic disorders such as FVIII deficiency (hemophilia A), FIX deficiency (hemophilia B), FVII deficiency, FX deficiency, FXI deficiency, FII deficiency, vWF deficiency, acquired antibodies against these coagulation factors, fibrinolysis abnormalities, platelets abnormalities, disseminated intravascular coagulation and any pathology associated with a combination of these deficiencies or abnormalities.

When used with an anticoagulant, the mutated antithrombin of the invention can be used for the preparation of a drug intended for the prevention or treatment of thrombotic disorders such as pulmonary embolism, deep vein thrombosis, myocardial infraction, unstable angina, stroke, disseminated intravascular coagulation.

In an advantageous embodiment, the present invention relates to the use of the above defined mutated antithrombin, wherein said mutated antithrombin comprises at least one mutation within the region from the amino acid at position 380 to the amino acid at position 400, particularly within the region from the amino acid at position 380 to the amino acid at position 397, particularly within the region from the amino acid at position 390 to the amino acid at position 394, in particular at position 393, the amino acid numbering referring to the antithrombin amino acid sequence represented by SEQ ID NO: 2, said mutation being a substitution, insertion or deletion.

The region from the amino acid at position 380 to the amino acid at position 400 is generally named “reactive center loop” (RCL). The residues in the RCL are numbered according to their positions relative to the scissile P1-P1′ bond (non-primed numbers towards the N-terminus and primed numbers towards the C-terminus of the serpin). In particular, the region stretching from P14 to P4′ (residues from 380 to 397) is complementary to the active site of its target protease. In particular, residues from P4 to P1′ (residues from 390 to 394) directly interact within protase catalytic groove. In particular, residue P1 (393) is crucial for protease inhibition.

The mutated antithrombins of the invention can comprise others mutations, outside of the region from the amino acid at position 380 to the amino acid at position 400, provided there is no change in the above-mentioned properties of the mutated antithrombins.

In an advantageous embodiment, the present invention relates to the use of the mutated antithrombin of the invention, wherein said mutated antithrombin further comprises at least one mutation at the glycosylation sites at the amino acid at position 96, 135, 155 or 192, in particular at position 135.

Glysosylation sites are necessary for antithrombin secretion when antithrombin is expressed in eukaryotic cells. However, removing one site does not impair antithrombin secretion while increases heparin binding. Indeed, glycosylation chains are involved in antithrombin-heparin binding.

In an advantageous embodiment, the present invention relates to the use such as defined above, of a mutated antithrombin, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of:

-   -   SEQ ID NO:4, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         substitution of the amino acid at position 393, by an Histidine         (His), or     -   SEQ ID NO:6, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         insertion of a Proline (Pro) between the amino acid at position         393 and the amino acid at position 394, or     -   SEQ ID NO:8, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         deletion of the amino acid at position 393, or     -   SEQ ID NO:10, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         deletion of the amino acid at position 394.

When there is one mutation, the mutated antithrombins above-mentioned are called single mutants.

In an advantageous embodiment, the present invention relates to the use such as defined above, of a mutated antithrombin, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of:

-   -   SEQ ID NO:14, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         substitution of the amino acid at position 393, by an Histidine         (His), and the substitution of the amino acid at position 135,         by a Glutamine (Gln), or     -   SEQ ID NO:16, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         insertion of a Proline (Pro) between the amino acid at position         393 and the amino acid at position 394, and the substitution of         the amino acid at position 135, by a Glutamine (Gln), or     -   SEQ ID NO:18, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         deletion of the amino acid at position 393 and at position 394,         or     -   SEQ ID NO:20, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         deletion of the amino acid at position 393 and the substitution         of the amino acid at position 135, by a Glutamine (Gln), or     -   SEQ ID NO:22, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         deletion of the amino acid at position 394 and the substitution         of the amino acid at position 135, by a Glutamine (Gln), or     -   SEQ ID NO:24, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:2, the         deletion of the amino acid at position 393 and at position 394,         and the substitution of the amino acid at position 135, by a         Glutamine (Gln).

When there are two or three mutations, the mutated antithrombins above-mentioned are called double and triple mutants respectively.

In an advantageous embodiment, the present invention relates to the use of the above-mentioned mutated antithrombin, wherein said mutated antithrombin comprises at least one mutation within the region from the amino acid at position 412 to the amino acid at position 432, particularly within the region from the amino acid at position 412 to the amino acid at position 429, particularly within the region from the amino acid at position 422 to the amino acid at position 426, in particular at position 425, the amino acid numbering referring to the antithrombin amino acid sequence comprising the signal peptide, represented by SEQ ID NO: 26, said mutation being a substitution, insertion or deletion.

The mutated antithrombins of the invention can comprise others mutations, outside of the region from the amino acid at position 412 to the amino acid at position 432, provided there is no change in the above-mentioned properties of the mutated antithrombins.

In an advantageous embodiment, the present invention relates to the use of the above-mentioned mutated antithrombin, wherein said mutated antithrombin further comprises at least one mutation at the glycosylation sites at the amino acid at position 128, 167, 187 or 224, in particular at position 167.

In an advantageous embodiment, the present invention relates to the use of the above-mentioned mutated antithrombin, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of:

-   -   SEQ ID NO:28, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         substitution of the amino acid at position 425, by an Histidine         (His), or     -   SEQ ID NO:30, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         insertion of a Proline (Pro) between the amino acid at position         425 and the amino acid at position 426, or     -   SEQ ID NO:32, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         deletion of the amino acid at position 425, or     -   SEQ ID NO:34, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         deletion of the amino acid at position 426.

In an advantageous embodiment, the present invention relates to the use of the above-mentioned mutated antithrombin, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of:

-   -   SEQ ID NO:38, said amino acid sequence comprising in the         sequence of antithrombin represented by SEQ ID NO:26, the         substitution of the amino acid at position 425, by an Histidine         (His), and the substitution of the amino acid at position 167,         by a Glutamine (Gln), or     -   SEQ ID NO:40, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         insertion of a Proline (Pro) between the amino acid at position         425 and the amino acid at position 426, and the substitution of         the amino acid at position 167, by a Glutamine (Gln), or     -   SEQ ID NO:42, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         deletion of the amino acid at position 425 and at position 426,         or     -   SEQ ID NO:44, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         deletion of the amino acid at position 425 and the substitution         of the amino acid at position 167, by a Glutamine (Gln), or     -   SEQ ID NO:46, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         deletion of the amino acid at position 426 and the substitution         of the amino acid at position 167, by a Glutamine (Gln), or     -   SEQ ID NO:48, said amino acid sequence comprising, in the         sequence of antithrombin represented by SEQ ID NO:26, the         deletion of the amino acid at position 425 and at position 426         and the substitution of the amino acid at position 167, by a         Glutamine (Gln).

The present invention also relates to a product comprising at least one mutated antithrombin having substantially no activity, in particular no anticoagulant activity, and at least one compound, in particular anticoagulant, as a combination product for a separate or sequential use in the prevention or treatment of hemorrhagic disorders and related pathologies resulting from side effects of said anticoagulant.

The expression <<for a separate or sequential use>> designates the use of the mutated antithromin between 5 minutes to 7 days after the last said anticoagulant administration, in particular 5 minutes to 72 hours.

The present invention relates to the above-mentioned product, wherein said mutated antithrombin has the ability to bind to the anticoagulant and to shift, in particular in vivo, the binding between plasma antithrombin and said anticoagulant.

The present invention relates to the above-mentioned product, wherein said mutated antithrombin has the ability to shift, in particular in vivo, the binding between plasma antithrombin and said anticoagulant and the ability to shift, in particular in vivo, the binding between said mutant of antithrombin and said anticoagulant.

The present invention relates to the above-mentioned product, wherein said mutated antithrombin has substantially lost factor Xa inhibitory activity and thrombin inhibitory activity.

According to an advantageous embodiment, the product according to the present invention is characterized in that the value of the dissociation equilibrium constant (Kd) of the complex resulting from the binding of said mutated antithrombin with said anticoagulant, at a given ionic strength, in particular at physiological ionic strength, is similar or lower than the value of the Kd of the complex between plasma antithrombin and said anticoagulant.

According to an advantageous embodiment, the product according to the present invention is characterized in that the value of the Kd of the complex resulting from the binding of said mutated antithrombin with said anticoagulant, at a given ionic strength, in particular at physiological ionic strength, is similar or lower than the value of the Kd of the complex between said mutant of antithrombin and said anticoagulant.

According to an advantageous embodiment, said anticoagulant is chosen among heparins, heparins derivatives, in particular unfractioned heparins, low molecular weight heparins, the anticoagulant pentasaccharide, (Fondaparinux), and its derivatives (Idraparinux: SANORG 34006), and heparinoids (Danaparoid sodium ORG 10172).

According to an advantageous embodiment, said coagulation disorders are among arterial or venous thrombotic disorders such as pulmonary embolism, deep vein thrombosis, myocardial infraction, unstable angina, stroke, disseminated intravascular coagulation and among hemorrhagic disorders such as FVIII deficiency (hemophilia A), FIX deficiency (hemophilia B), FVII deficiency, FX deficiency, FXI deficiency, FII deficiency, vWF deficiency, acquired antibodies against these coagulation factors, fibrinolysis abnormalities, platelets abnormalities, disseminated intravascular coagulation and any pathology associated with a combination of these deficiencies or abnormalities.

In an advantageous embodiment, the present invention relates to the product such as defined above, wherein said mutated antithrombin comprises at least one mutation within the region from the amino acid at position 380 to the amino acid at position 400, particularly within the region from the amino acid at position 380 to the amino acid at position 397, particularly at least one mutation within the region from the amino acid at position 390 to the amino acid at position 394, in particular at position 393, the amino acid numbering referring to the antithrombin III amino acid sequence represented by SEQ ID NO: 2, said mutation being a substitution, insertion or deletion.

In an advantageous embodiment, the present invention relates to the product such as defined above, wherein said mutated antithrombin further comprises at least one mutation at the glycosylation sites at the amino acid at position 96, 135, 155 or 192, in particular at position 135.

In an advantageous embodiment, the present invention relates to the product such as defined above, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of:

-   -   SEQ ID NO:4,     -   SEQ ID NO:6,     -   SEQ ID NO:8, or     -   SEQ ID NO:10.

In an advantageous embodiment, the present invention relates to the product such as defined above, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of:

-   -   SEQ ID NO:14,     -   SEQ ID NO:16,     -   SEQ ID NO:18,     -   SEQ ID NO:20,     -   SEQ ID NO:22, or     -   SEQ ID NO:24.

In another advantageous embodiment, the present invention relates to the product such as defined above, wherein said mutated antithrombin comprises at least one mutation within the region from the amino acid at position 412 to the amino acid at position 432, particularly within the region from the amino acid at position 412 to the amino acid at position 429, particularly at least one mutation within the region from the amino acid at position 422 to the amino acid at position 426, in particular at position 425, the amino acid numbering referring to the antithrombin amino acid sequence comprising the signal peptide, represented by SEQ ID NO: 26, said mutation being a substitution, insertion or deletion.

In an advantageous embodiment, the present invention relates to the product such as defined above, wherein said mutated antithrombin further comprises at least one mutation at the glycosylation sites at the amino acid at position 128, 167, 187 or 224, in particular at position 167.

In an advantageous embodiment, the present invention relates to the product such as defined above, wherein said mutated antithrombin is chosen from the group consisting of:

-   -   SEQ ID NO:28,     -   SEQ ID NO:30,     -   SEQ ID NO:32 or     -   SEQ ID NO:34.

In an advantageous embodiment, the present invention relates to the product such as defined above, wherein said mutated antithrombin is chosen from the group consisting of:

-   -   SEQ ID NO:38,     -   SEQ ID NO:40,     -   SEQ ID NO:42,     -   SEQ ID NO:44,     -   SEQ ID NO:46, or     -   SEQ ID NO:48.

The present invention also relates to the use of a mutated antithrombin having substantially no activity, in particular no anticoagulant activity, without anticoagulant, for the preparation of a drug intended for the prevention or treatment of pathologies linked to or associated with coagulation disorders.

When used without association with an anticoagulant, the mutated antithrombin of the invention can be used for the preparation of a drug intended for the prevention or treatment of hemorrhagic disorders such as FVIII deficiency (hemophilia A), FIX deficiency (hemophilia B), FVII deficiency, FX deficiency, FXI deficiency, FII deficiency, vWF deficiency, acquired antibodies against these coagulation factors, fibrinolysis abnormalities, platelets abnormalities, disseminated intravascular coagulation and any pathology associated with a combination of these deficiencies or abnormalities.

In an advantageous embodiment, the present invention relates to the use such as defined above, of a mutated antithrombin having substantially no activity, in particular no anticoagulant activity, without anticoagulant, for the preparation of a drug intended for the prevention or treatment of hemorrhagic disorders.

The present invention also relates to a mutated antithrombin, which contains at least an amino acid insertion between the amino acid at position 393 and the amino acid at position 394, in the sequence of antithrombin represented by SEQ ID NO:2, in particular an insertion of a Proline (Pro), and in particular mutated antithrombin represented by SEQ ID NO: 6.

The present invention also relates to a mutated antithrombin, which contains at least an amino acid insertion between the amino acid at position 425 and the amino acid at position 426, in the sequence of antithrombin represented by SEQ ID NO:26, in particular an insertion of a Proline (Pro), and in particular mutated antithrombin represented by SEQ ID NO: 30.

The present invention also relates to a mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 393, in the sequence of antithrombin represented by SEQ ID NO:2, in particular mutated antithrombin represented by SEQ ID NO: 8.

The present invention also relates to a mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 425, in the sequence of antithrombin represented by SEQ ID NO:26, in particular mutated antithrombin represented by SEQ ID NO: 32.

The present invention also relates to a mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 394, in the sequence of antithrombin represented by SEQ ID NO:2, in particular mutated antithrombin represented by SEQ ID NO: 10.

The present invention also relates to a mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 426, in the sequence of antithrombin represented by SEQ ID NO:26, in particular mutated antithrombin represented by SEQ ID NO: 34.

The present invention also relates to a mutated antithrombin, which contains at least two mutations:

-   -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:2, the substitution of the amino acid         at position 393, by an Histidine (His), the second mutation         being the substitution of the amino acid at position 135, by a         Glutamine (Gln), said mutated antithrombin being in particular         represented by SEQ ID NO: 14,     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:2, the insertion of a Proline (Pro)         between the amino acid at position 393 and the amino acid at         position 394, the second mutation being the substitution of the         amino acid at position 135, by a Glutamine (Gln), said mutated         antithrombin being in particular represented by SEQ ID NO: 16,         or     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:2, the deletion of the amino acid at         position 393, the second mutation being the deletion of the         amino acid at position 394, said mutated antithrombin being in         particular represented by SEQ ID NO: 18, or     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:2, the deletion of the amino acid at         position 393, the second mutation being the substitution of the         amino acid at position 135, by a Glutamine (Gln), said mutated         antithrombin being in particular represented by SEQ ID NO: 20,         or     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:2, the deletion of the amino acid at         position 394, the second mutation being the substitution of the         amino acid at position 135, by a Glutamine (Gln), said mutated         antithrombin being in particular represented by SEQ ID NO: 22,         or     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:2, the deletion of the amino acid at         position 393, the second mutation being the deletion of the         amino acid at position 394, and the third mutation being         substitution of the amino acid at position 135, by a Glutamine         (Gln), said mutated antithrombin being in particular represented         by SEQ ID NO: 24.

The present invention also relates to a mutated antithrombin, which contains at least two mutations:

-   -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:26, the substitution of the amino acid         at position 425, by an Histidine (His), the second mutation         being the substitution of the amino acid at position 167, by a         Glutamine (Gln), said mutated antithrombin being in particular         represented by SEQ ID NO: 38,     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:26, the insertion of a Proline (Pro)         between the amino acid at position 425 and the amino acid at         position 426, the second mutation being the substitution of the         amino acid at position 167, by a Glutamine (Gln), said mutated         antithrombin being in particular represented by SEQ ID NO: 40,         or     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:26, the deletion of the amino acid at         position 425, the second mutation being the deletion of the         amino acid at position 426, said mutated antithrombin being in         particular represented by SEQ ID NO: 42, or     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:26, the deletion of the amino acid at         position 425, the second mutation being the substitution of the         amino acid at position 167, by a Glutamine (Gln), said mutated         antithrombin being in particular represented by SEQ ID NO: 44,         or     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:26, the deletion of the amino acid at         position 426, the second mutation being the substitution of the         amino acid at position 167, by a Glutamine (Gln), said mutated         antithrombin being in particular represented by SEQ ID NO: 46,         or     -   the first mutation being, in the sequence of antithrombin         represented by SEQ ID NO:26, the deletion of the amino acid at         position 425, the second mutation being the deletion of the         amino acid at position 426, and the third mutation being         substitution of the amino acid at position 167, by a Glutamine         (Gln), said mutated antithrombin being in particular represented         by SEQ ID NO: 48.

The present invention also relates to a nucleotide sequence encoding a mutated antithrombin as defined above, in particular nucleotide sequences chosen in the group consisting of SEQ ID NO: 5, 7, 9, 13, 15, 17, 19, 21, 23, 29, 31, 33, 37, 39, 41, 43, 47.

The present invention also relates to a pharmaceutical composition comprising as active ingredient a mutated antithrombin as defined above, in combination with a pharmaceutical acceptable vehicle, in particular a mutated antithrombin of SEQ ID NO: 6, 8, 10, 14, 16, 18, 21, 22, 24, 30, 32, 34, 38, 40, 42, 44, 46, 48.

The present invention also relates to a pharmaceutical composition, comprising as active ingredient a mutated antithrombin, of SEQ ID NO:4, 12, 28 or 36 in combination with a pharmaceutical acceptable vehicle.

FIGURES

FIG. 1: preparation of shuttle vector carrying full length antithrombin cDNA.

2 μg of pENTR vector containing truncated AT cDNA (lane 1 and 2), pCMV6 vector containing full length AT cDNA (lane 3 and 4) or shuttle vector pENTR-AT (lane 5 and 6) are loaded on 1% agarose gel before (lane 1, 3, 5) or after (lane 2, 4, 6) complete digestion by both SacII and StuI. Molecular weight standard sizes are indicated on the left hand of the figure and expressed in base pair (bp). SacII/StuI digestion of pCMV6 vector containing full length AT cDNA releases a 1182 bp band corresponding to full length AT cDNA cloned into pENTR vector isolated from SacII/StuI digestion of pENTR vector containing truncated AT cDNA. After ligation of these two fragments the final product is effectively recircularized and shows the expected profile for pENTR-AT after SacII/StuI digestion.

FIG. 2: Characterization of the AT expression vector.

2 μg of pCDNA 3.2 vector (lane 1, 2, 3), pCDNA 3.2 vector containing full length AT cDNA (lane 4, 5, 6) or pCDNA 3.2 vector containing full length AT-N135Q-Pro394 cDNA (lane 7, 8, 9) are loaded on 1% agarose gel before endonucleases treatment (lane 1, 4, 7), after cleavage by StuI (lane 2, 5, 8), or after complete cleavage by both SacII and StuI (lane 3, 6, 9). Molecular weight standard sizes are indicated on the right hand of the figure and expressed in base pair (bp). There is one SacII and one StuI cleavage site in pCDNA 3.2 vector at position 3189 and 4329 respectively. Therefore cleavage of pCDNA 3.2 by StuI only leads to linearization of the vector (7711 bp band) whereas cleavage of pCDNA 3.2 by both StuI and SacII cuts the vector in two fragments (6571 bp and 1140 bp). Substitution of 912-3174 fragment by AT cDNA fragment (1448 bp) into pCDNA 3.2 by recombination introduces one more SacII site at position 1070 and one more StuI site at position 2252 into pcDNA-AT. Then cleavage of pCDNA-AT by StuI gives two fragments (5634 bp and 1263 bp) and cleavage by both endonucleases gives 4 fragments (4452 bp, 1182 bp, 1140 and 123 bp). The same is true for —N135Q-Pro394 cDNA.

FIG. 3: Clone screening for secretion of recombinant AT in cell culture media.

For each clone isolated after transfection with pCDNA-AT (clone 1 to 5 in lane 1 to 5, respectively), pCDNA-AT-N135Q (clone 1 to 4 in lane 6 to 9, respectively), or with pCDNA-AT-N135Q-Pro394 (clone 1 to 5 in lane 10 to 14, respectively), 30 μl of conditioned media harvested after 24 hours contact with cells are analysed by western-blotting in denaturing condition for their ability to secrete full length recombinant antithrombin. For each clone a single band of variable intensity corresponding to recombinant antithrombin can be seen. Recombinant wild type antithrombin migrate at the same level than control antithrombin purified from plasma (lane 15, 150 ng/lane) and expression level is estimated around 2 mg/L according to band intensity measurement. For wt-AT, clone 4 is chosen for expansion into cell factory, since the level of expression seems to be slightly higher than the others. Mutant AT-N135Q and AT-N135Q-Pro394 migrate just below control antithrombin purified from plasma (lane 15) confirming the loss of a glycosylation site due to substitution N135Q. The stable expression clones selected for large scale protein production are clones 1 for both AT-N135Q and AT-N135Q-Pro394.

FIG. 4: Integrity and purity of recombinant antithrombin.

To verify integrity and purity of recombinant antithrombin after heparin affinity purification and ion exchange concentration, 2 μg of AT-N135Q-R393H (lane 1), AT-N135Q-Pro394 (lane 2) or control plasma antithrombin (lane 3) are analyzed by SDS-PAGE followed by coomassie staining. As expected, the two mutated antithrombins migrate at molecular weight slightly lower than plasma antithrombin because of loss of a glycosylation site (substitution N135Q) and they show a single band pattern with band intensity corresponding to quantity loaded on the gel (based on absorbance estimation). Then recombinant antithrombin appears pure and can be tested for its anticoagulant properties and affinity for heparin derivatives. Molecular weight standard sizes are presented on the right hand of the figure and expressed in kiloDalton (KD)

FIG. 5: Anti-factor Xa activity of plasmatic or mutated antithrombins at saturating pentasaccharide concentration.

FIG. 5 a: Plasma AT (black square (or black rectangle): 80 nM, hollow square: 40 nM, Black circle: 20 nM, hollow circle: 10 nM) is tested for its ability to inhibit chromogenic substrate S2765 (200 μM) hydrolysis by FXa (1 nM) in the presence of pentasaccharide (1 μM or 1.73 mg/L) in continuous assay. Time expressed in second is plotted in abscissa; absorbence at 405 nm is plotted in ordinate.

FIG. 5 b: FIG. 5 a substrate hydrolysis curves are fitted using equation 3 to determine the kinetic rate constant (k). This constant k is thus plotted as the function of AT concentration and fitted using equation 4 to determine inhibition rate constant kon. The plasma antithrombin concentration (nM) is plotted in abscissa; the kinetic rate constant (k) expressed in s⁻¹ is plotted in ordinate.

FIG. 6: Anti-factor Xa activity of mutated antithrombins in discontinuous assay AT-N135Q-R393H and AT-N135Q-Pro394 are tested for their ability to inhibit FXa activity in discontinuous assay. In a first time AT-N135Q-R393H (black square: 200 nM) or AT-N135Q-Pro394 (hollow square: 2.5 μM) are incubated with FXa (20 nM) in the presence of pentasaccharide (10 μM or 17.3 mg/L) over a period of time from 0 to 120 min (for AT-N135Q-R393H) or from 0 to 1400 min (for AT-N135Q-Pro394). In a second time FXa residual activity is measured by adding 190 μl of S2765 (200 μM) to 10 μl of previous mixture. Initial rate of substrate hydrolysis is then plotted as the function of incubation time with inhibitor and curves are fitted with equation 1 to determine kon (gray lines). Time expressed in minute is plotted in abscissa; substrate hydrolysis rate expressed in OD/s⁻¹ is plotted in ordinate.

FIG. 7: AT can compete with plasma AT for pensaccharide binding

AT-N135Q-R393H and AT-N135Q-Pro394 are tested in Rotachrom® heparin assay to establish residual pentassacharide activity. Mutated antithrombins in a 1/1, 2/1 and 4/1 ratio compared to plasma antithrombin, are added to human plasma pool over-dosed with pentasaccharide (concentration of 3 μg/ml).

Bars 1, 3, 5 and 7 represent results with AT-N135Q-R393H and Bars 2, 4, 6 and 8 represent results with AT-N135Q-Pro394. Bars 1 and 2 are control bars without mutated antithrombin. Bars 3 and 4 represent mutated antitrombin ratio 1/1 to plasma antithrombin. Bars 5 and 6 represent mutated antitrombin ratio 2/1 to plasma antithrombin. Bars 7 and 8 represent mutated antitrombin ratio 4/1 to plasma antithrombin.

FIG. 8: AT can compete with plasma AT for pensaccharide binding when incubated on HUVEC surface.

AT-N135Q-R393H and AT-N135Q-Pro394 are tested in Rotachrom® heparin assay to establish residual pentassacharide activity. Mutated antithrombins in a 1/1, 2/1 and 4/1 ratio compared to plasma antithrombin, are added to human plasma pool over-dosed with pentasaccharide (concentration of 3 μg/ml) and incubated on HUVEC surface.

Bars 1, 3, 5 and 7 represent results with AT-N135Q-R393H and Bars 2, 4, 6 and 8 represent results with AT-N135Q-Pro394. Bars 1 and 2 are control bars without mutated antithrombin. Bars 3 and 4 represent mutated antitrombin ratio 1/1 to plasma antithrombin. Bars 5 and 6 represent mutated antitrombin ratio 2/1 to plasma antithrombin. Bars 7 and 8 represent mutated antitrombin ratio 4/1 to plasma antithrombin.

EXPERIMENTAL PART

In order to produce a mutated antithrombin having lost anticoagulant activity, in particular factor Xa and IIa inhibitory activity, and able to bind to heparin and to the pentasaccharide, different types of mutations have been contemplated and particularly, mutations within the reactive center loop (region from the amino acid 380 to amino acid 400), mutations within an exosite region remote from the loop accessible for proteinase interaction (Chuang Y J, Swanson R, Raja S M, Olson S T. Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin J Biol Chem. 2001; 276:14961-71) and mutations within a consensus sequence of glycosylation (amino acids 135 to 137 and 155 to 157) (Fan B, Crews B C, Turko I V, Choay J, Zettlmeissl G, Gettins P. Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure J Biol Chem. 1993; 268:17588-96).

Several mutations have been carried out to obtain mutated antithrombins, and in particular:

deletion within the reactive loop of the antithrombin, in the region P4-P4′ in order to eliminate antithrombin inhibitory activity toward any coagulant proteases such as FXa and FIIa,

substitution of amino acids within the region P4-P4′ (Ala 391-Asn 396) of the reactive loop, and in particular the substitution of the amino acid at position 393 (Arg) by an Histidine,

insertion of a Proline between the amino acid at position 393 and the amino acid at position 394, and

substitution of amino acids within the region of glycosylation of the antithrombin, and in particular the substitution of the amino acid at position 135 (Asp) by a Glutamine, in order to increase the affinity with heparin and pentasaccharide.

Material and Methods

I/ Preparation of Mutated Antithrombins

Preparation of Shuttle Vector pENTR Carrying Full Length Antithrombin cDNA (pENTR-AT):

The antithrombin cDNA sequence initially cloned into pENTR vector (Invitrogen, HORF clone reference IOH14497) is found to be truncated and has to be replaced by the full length antithrombin sequence, cloned into pCMV6 (Origene, reference TC110831). The plasmid pCMV6 containing full length antithrombin cDNA is digested by both SacII and StuI endonucleases. The 1182 base pairs fragment is isolated on 1% agarose gel and purified using the QIAquick Gel Extraction Kit. This 1182 base pairs fragment, corresponding to the SacII-StuI fragment of antithrombin cDNA, is ligated into pENTR vector (2760 bp) also linearized by SacII and StuI and recover as described above. Result of this cassette exchange is verified by electrophoresis on 1% agarose gel (FIG. 1) and sequencing.

Mutagenesis on Antithrombin cDNA:

The resulting plasmid pENTR carrying cDNA encoding for wild type antithrombin (pENTR-ATwt) is used as a template for further mutagenesis by PCR using the QuickChange II Site-Directed Mutagenesis Kit according to the manufacturer recommendations (Stratagene). The wild type antithrombin has the same amino acid sequence as the plasma antithrombin but is produced under a recombinant form. Mutagenic primers (table 1) are used to introduce a codon for Glutamine in place of codon for Arginine 135 for production of plasmid pENTR-AT-N135Q. Single amino acid substitution of Arginine 393 by an Histidine (R393H), insertion of a Proline between Arginine 393 and Serine 394 (Pro394), or deletion of Arginine 393 (ΔR393), Serine 394 (ΔS394) or both Arginine 393 and Serine 394 (ΔR393S394) are introduced by PCR using the QuickChange II Site-Directed Mutagenesis Kit with pENTR-ATwt as template and mutagenic primers as described in table 1. The same couples of mutagenic primers are used in PCR reaction with pENTR-AT-N135Q as template to prepare plasmids carrying cDNA encoding for double-mutant and triple-mutant antithrombin N135Q-R393H, N135Q-Pro394, N135Q-ΔR393, N135Q-45394 and N135Q-ΔR393S394 respectively. Then the integrity of each variant cassette that is to say cDNA encoding for the double-mutant or triple-mutant antithrombin above mentioned is established by DNA sequencing.

Cassette Exchange Between Shuttle Vector and Expression Vector:

All the cDNAs described above encoding for antithrombin, single antithrombin mutants or double antithrombin mutants are transferred from shuttle vector pENTR into eucaryote expression vector pCDNA 3.2 by recombination using Gateway LR Clonase II Enzyme Mix (“Gateway Technology” developed by Invitrogen). The final expression constructs are verified by electrophoresis on 1% agarose gel and sequencing again before transfection (FIG. 2).

Transfection of Eucaryote Cells and Protein Production:

Plasmid constructs resulting of previous recombination named pCDNA-ATwt, pCDNA-AT-N135Q, pCDNA-AT-R393H, pCDNA-AT-Pro394, pCDNA-AT-ΔR393, pCDNA-AT-ΔS394, pCDNA-AT-ΔR393S394, pCDNA-AT-N135Q-R393H, pCDNA-AT-N135Q-Pro394, pCDNA-AT-N135Q-ΔR393, pCDNA-AT-N135Q-ΔS394, and pCDNA-AT-N135Q-ΔR393S394, respectively are used for transfection of modified human embryonic kidney cells (HEK-293) or baby hamster kidney cells (BHK-21). Cells are grown in “Dulbeco's Modified Eagle's Medium/F-12” containing 2 mM L-Glutamine, 100 U/ml penicillin, 100 μg/ml Streptomycin and 5% foetal bovine serum (Invitrogen) and approximately 10⁶ cells are transfected with 20 μg of DNA by calcium-phosphate coprecipitation (Sambrook et al. Molecular cloning: A laboratory manual, 2^(nd) edition, page 16.33). The stable expression cell lines are selected by G418 (during clones selection, G418 concentration is 0.8 mg/ml in cell culture media and then decreased to 0.4 mg/ml to maintain selection pressure during clones amplification) and screened for antithrombin secretion by ELISA, using mouse monoclonal antibody anti antithrombin as capture antibody and Horse Radish Peroxydase conjugate sheep polyclonal antibody anti antithrombin as detecting antibody (BioAssay™ ELISA Kit (EUROMEDEX). The integrity of secreted antithrombin is established by western blotting using sheep monoclonal antibody anti antithrombin and Horse Radish Peroxydase conjugate donkey polyclonal antibody anti sheep (The Binding Site, UK) (FIG. 3). For each mutated antithrombin, a single stable expression clone is expanded into “cell factories nunclon” (Nunc) and large scale protein production is conducted with 300 μl/cm², (as recommended by manufacturer, the minimum volume suitable for a 6320 cm² culture area cell factory is 2 liters) of <<Dulbeco's Modified Eagle's Medium/F-12” containing 2 mM L-Glutamine, 100 U/ml penicillin, 100 μg/ml Streptomycin and 5 μg/ml Insulin/Transferrin/Selenium (Invitrogen). Conditioned media, harvested daily, are centrifuged for 15 min at 3000 g at 4° C., treated with 5 mM benzamidine, 5 mM EDTA and stored at −20° C.

Protein Purification:

Conditioned media are thawed, pooled, salt concentration adjusted to 0.4 M NaCl and then applied on an heparin immobilized column (Hitrap Heparin 5 ml or Heparin-sepharose CL6B 50 ml, GE Biological) equilibrated with 10 mM Tris, or with 20 mM phosphate buffer, 0.4 M NaCl and 0.1 mM EDTA, pH 7.4. The bound proteins are eluted in the same buffer with a gradient from 0.4 M to 2 M NaCl. The fractions eluted from 0.8 M NaCl and more contained only wild type antithrombin or mutated antithrombins with high heparin affinity. Mutated antithrombins carrying substitution of asparagin 135 by a glutamin (AT-N135, ATN135Q-R393H, AT-N135Q-Pro394, AT-N135Q-ΔR393, AT-N135Q-ΔS394, AT-N135Q-ΔR393S394) are eluted from affinity column at higher ionic strength than wild type antithrombin, confirming that destruction of glycosylation site at position 135 increases affinity of antithrombin for heparin (about 90% of mutanted antithrombin carrying substitution N135Q is eluted between 1 and 1.4 M NaCl compared to wild type antithrombin which 90% is eluted between 0.8 and 1.2 M NaCl). The collected fractions are pooled and the salt concentration is decreased either by over night dialysis against 10 mM Tris, or 20 mM phosphate, and 0.1 M NaCl, pH 7.4 at 4° C. or applied on an HiPrep 26/10 desalting column equilibrated with 10 mM Tris, or 20 mM phosphate, and 0.1 M NaCl, pH 7.4. The antithrombin is then concentrated by ion exchange chromatography using a “Resource Q” 1 ml column (GE, Biological) equilibrated with 10 mM Tris, or 20 mM phosphate, and 0.1 M NaCl, pH 7.4 and eluted in the same buffer with a NaCl gradient from 0.1 mM to 0.5 M or 20 mM to 0.5 M. The antithrombin concentration in each elution fraction is estimated by absorbance at 280 nM with an absorption coefficient ε=0.65 g⁻¹.l.cm⁻¹ and the integrity of purified wild type antithrombin or mutated antithrombins is tested by western blotting using the same couple of antibodies as described before, and electrophoresis on 10% acrylamide/bisacrylamide gel in native and denaturing conditions followed by coomassie brilliant blue R-250 staining (FIG. 4). Then, the antithrombin preparation is aliquoted and stored at −80° C. before use for functional assay. The same procedure is used to purify plasma antithrombin (used as internal reference) from human plasma.

TABLE 1 Sens Mutation Sequence forward N135Q GCCGACTCTATCGAAAAGCCC AGAAATCCTCCAAGTTAGTG reverse N135Q CACTAACTTGGAGGATTTCTG GGCTTTTCGATAGAGTCGGC forward R393H GTTGTGATTGCTGGCCATTCGCTA AACCCCAAC reverse R393H GTTGGGGTTTAGCGAATGGCCAGC AATCACAAC forward Pro394 GTTGTGATTGCTGGCCGTCCATCGCTAAA CCCCAAC reverse Pro394 GTTGGGGTTTAGCGATGGACGGCCAGCAA TCACAAC forward ΔR393- GCTGTTGTGATTGCTGGCCTAAACCCCAA S394 CAGGGTG reverse ΔR393- CACCCTGTTGGGGTTTAGGCCAGCAATC S394 ACAACAGC forward ΔR393 CTGTTGTGATTGCTGGCTCGCTAAAC CCCAACAG reverse ΔR393 CTGTTGGGGTTTAGCGAGCCAGCAAT CACAACAG forward ΔS394 TGTGATTGCTGGCCGTCTAAACCC CAACAGGG reverse ΔS394 CCCTGTTGGGGTTTAGACGGCCA GCAATCACA

II/ In Vitro Characterization of the Mutated Antithrombins

Characterization of the mutated antithrombins aims at

a) demonstrating that, in a purified system, the following mutated antithrombins: AT-R393H, AT-Pro394, AT-ΔR393-S394, AT-ΔR393, AT-ΔS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135Q-ΔR393-S394, AT-N135Q-ΔR393 and AT-N135Q-ΔS394 exhibit a negligible anti FXa and anti FIIa activity compared with that of wild type antithrombin (AT-wt) in the presence or absence of heparin or pentasaccharide,

b) demonstrating that, in plasma, the following mutated antithrombins AT-R393H, AT-Pro394, AT-ΔR393-S394, AT-ΔR393, AT-ΔS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135Q-ΔR393-S394, AT-N135Q-ΔR393, AT-N135Q-ΔS394, can compete with plasma antithrombin for heparin or pentasaccharide binding and produce a significant decrease in anti factor Xa or anti factor IIa activity in plasma.

a) Anti Factor Xa Inhibitory Activity of the Mutated Antithrombins in a Purified System

The kinetic assays for antithrombin inhibition of factor Xa (FXa, Kordia) are performed in ‘kinetic’ buffer (Hepes, 20 mM phosphate, pH 7.4, 0.15 M NaCl, 0.1% PEG 8000 and 1 mg/ml bovine serum albumin) in pseudo first order conditions. Briefly, factor Xa is incubated with an excess of tested antithrombins (corresponding to plasma antithrombin, or wild type antithrombin or mutated antithrombins and varying in each assay realized) in the presence or absence of pentasaccharide (Fondaparinux sodique, Arixtra®, GlaxoSmithKline) and the factor Xa residual activity is measured as a function of time. The pentasaccharide is added in excess in the reaction media so that every tested antithrombin is bound to the pentasaccharide. In the absence of pentasaccharide, polybrene is added in order to neutralize any sulfated glycosaminoglycan that may be present in the reaction media. The residual factor Xa activity is measured as the increase in absorbance at 405 nm resulting from cleavage of the chromogenic substrate (S2765 or S2222, Chromogenix) using a microplate reader (Dynatech MR 5000). The analysis of the data is performed using the GraphPad Prism version 3 software. Absorbance recording is continuous or discontinuous according to the expected inhibition rate constant (kon).

The inhibition rate constant (kon) is the second order rate constant given in M⁻¹.s⁻¹ which define the velocity of stable complex formation between protease and antithrombin (higher is the kon value, faster will the complex be established).

When the expected kon is lower than 10⁴ M⁻¹.s⁻¹ the discontinuous method is used. Factor Xa (0.02 to 1 μM, or 0.1 to 1 μM) is incubated with tested antithrombins (0.2 to 10 μM, or 1 to 10 μM) in the presence of pentasaccharide (10 to 100 μM) or polybrene (100 μg/ml) in a final volume of 10 μl for 10 seconds to 5 hours, or 10 seconds to 24 hours. At the end of this incubation period, 190 μl of kinetic buffer containing 200 μM substrate is added and absorbance at 405 nm is recorded.

The kinetic rate constant (k) is estimated by fitting the substrate hydrolysis initial rate curve to equation (1) using non-linear regression with v0 and v∞ being the substrate hydrolysis rate at time to or t∞ respectively.

vt=(v0+v∞).exp(−k.t)   (1)

The inhibition rate constant (kon) is calculated from the rate constant (k) using equation (2) where AT is the tested antithrombin concentration.

k=AT.kon   (2)

When the expected kon is higher than 10⁴ M⁻¹.s⁻¹′ the continuous method is used. The tested antithrombin (0.01 to 1 μM, or 0.1 to 1 μM) is incubated with the substrate (200 μM) in the presence of pentasaccharide or polybrene in a final volume of 100 μL, or 190 μl, and the reaction is started by addition of 100 μL, or 10 μL, of factor Xa (2 nM or 10 nM). The rate constant (k) is obtained by fitting the substrate hydrolysis curve to equation (3) or (3′) using non-linear regression analysis where A0 is the absorbance at t0, and vi and vs are respectively the initial and final rates of substrate hydrolysis in the absence of tested antithrombin.

A405=A0+vi.(1−exp(−k.t))/k.   (3)

Equation (3′) is also used, particularly in presence of plasma.

A405=A0+vs*t+(vi−vs)*(1−exp(−k.t))/k.   (3′)

The inhibition rate constant (kon) is calculated from the kinetic constant (k) using equation (4) that takes into account the competitive effect of the substrate, with S being the initial substrate concentration, Km, the Michaelis constant for factor Xa-substrate interaction, AT the tested antithrombin concentration.

k=(kon/(1+S/Km)).[AT]  (4)

Mutated antithrombins and wild type AT inhibitory activity are measured in the same conditions and compared with plasma AT inhibitory activity.

The results with or without pentasaccharide are:

-   -   wild type antithrombin and AT-N135Q factor Xa inhibitory         activity is similar to plasma antithrombin factor Xa inhibitory         activity,     -   factor Xa inhibitory activity of the following mutated         antithrombins: AT-R393H, AT-Pro394, AT-ΔR393-S394, AT-ΔR393,         AT-ΔS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135Q-ΔR393-S394,         AT-N135Q-ΔR393, AT-N135Q-ΔS394, is negligible compared with wild         type antithrombin factor Xa inhibitory activity.

For example, inhibition rate constant (kon) of plasma AT for factor Xa in the presence of saturating amount of pentassacharide is estimated using continuous method (FIGS. 5 a and 5 b). A value of 2.52×10⁵ M⁻¹.s⁻¹ is found which is comparable to published values (Olson S T, Björk I, Sheffer R, Craig P A, Shore J D, Choay J., J Biol Chem. 1992 Jun. 25; 267 (18): 12528-38, “Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.) Using this method AT-N135Q-R393H and AT-N135Q-Pro394 are found to be slow factor Xa inhibitors, even in the presence of saturating pentassacharide concentration. Thus, discontinuous method was performed to evaluate kon values for factor Xa inhibition by AT-N135Q-R393H and AT-N135Q-Pro394 in the presence of pentasaccharide (FIG. 6). AT-N135Q-R393H anticoagulant activity is largely reduced whereas AT-N135Q-Pro394 is almost devoided of anti-factor Xa activity. AT-N135Q-R393H and AT-N135Q-Pro394 kon values are estimated at 4415 M⁻¹.s⁻¹ and 33 M⁻¹.s⁻¹, respectively, which is 95 times and at least 7600 times lower than plasma AT.

b) Anti Factor Xa Inhibitory Activity of the Mutated Antithrombins in Human Plasma

Anti factor Xa inhibitory activity of mutated antithrombins AT-R393H, AT-Pro394, AT-ΔR393-S394, AT-ΔR393, AT-ΔS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135Q-ΔR393-S394, AT-N135Q-ΔR393, AT-N135Q-ΔS394 is measured in pentasaccharide containing plasma. The plasma concentrations of pentasaccharides are defined on the basis on those measured in patients treated by the pentasaccharide under the conditions of a regular (curative) use of this molecule or in patients presenting an overdose of this drug, knowing that the optimal equilibrium concentration is 1.20-1.26 mg/L and the minimal equilibrium concentration is 0.46-0.62 mg/L during a curative pentasaccharide treatment (monograph, Arixtra®).

The anti factor Xa assay is performed using the Rotachrom® heparin method (Stago) adaptated for absorbance reading on a microplate reader (Dynatech MR 5000), or a STA analyser (Stago).

The procedure consists in mixing 30 μl of pentasaccharide containing plasma sample diluted four times in kinetic buffer, with 75 μl of chromogenic substrate (1 mM, provided in Rotachrom® heparin kit). After 4 min incubation, 75 μl of bovine factor Xa (24 nM, Rotachrom® heparin kit) is added to the plasma-substrate mixture. The factor Xa activity is measured a 405 nm absorbance increase, resulting from cleavage of the chromogenic substrate, using a microplate reader (Dynatech MR 5000). The rate constant (k), proportional to anti-factor Xa activity, is obtained by fitting the substrate hydrolysis curve to equation (3) or (3′).

Firstly, the assay is calibrated in citrated (0.105 M) normal human plasma containing pentasaccharide concentrations ranging from 0 to 3 mg/L. A standard curve is generated and anti-factor Xa activity is expressed as mg of pentasaccharide per liter of plasma.

Secondly, to evaluate the anti factor Xa inhibitory activity of mutated antithrombins, the mutated antithrombin (0-0.6 g/L, preferably 0.15-0.6 g/L, final concentration, in kinetic buffer) is pre-incubated with an equal volume of pentasaccharide (0-3 mg/L, preferably 1-3 mg/L) containing plasma samples, during 30 min at 37° C.

Under these conditions, the mutated antithrombin concentration range from one to four times that of the plasma antithrombin level, which is compatible with a therapeutic use of antithrombin. Indeed, very high doses of mutated antithrombin, with a 5 fold increase in plasma antithrombin level, have already been used during clinical trials without side effects (Leitner J M, Firbas C, Mayr F B, Reiter R A, Steinlechner B, Jilma B., Recombinant human antithrombin inhibits thrombin formation and interleukin 6 release in human endotoxemia. Clin Pharmacol Ther. 2006 January; 79(1):23-34). Anti factor Xa inhibitory activity is then evaluated as previously described.

Finally to evaluate effect of mutated antithrombin on anti-factor Xa activity in conditions as close as possible to in vivo conditions, Rotachrom® heparin method is used to test anti-factor Xa activity in plasma pre-incubated with mutated antithrombin (0-0.6 g/L, preferably 0.15-0.6 g/L in kinetic buffer), during 30 min at 37° C. on human umbilical vein endothelial cells (HUVEC) in culture. Cells are inoculated at 20 000 cells/cm² in 96 well plate and grown to confluence for 24 hours in EGM-2 media (Clonetics, Cambrex). After two wash with phosphate buffer salin (Gibco, Invitrogen) cells are starved for 4 hours in EBM-2 media (Clonetics, Cambrex) and then washed again twice in phosphate buffer salin to prevent excess contamination with heparin present in the culture medium. HUVEC coated wells are then used for 30 min incubation of pentasaccharide containing plasma-mutated antithrombin mixture (125 μl/cm²) before Rotachrom® heparin assay

It is demonstrated that the mutated antithrombins compete with plasma antithrombin for pentasaccharide binding and are able to decrease significantly the plasma anti-Xa activity.

In the condition of the assay, addition of AT-R393H, AT-Pro394, AT-ΔR393-S394, AT-ΔR393, AT-ΔS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135Q-ΔR393-S394, AT-N135Q-ΔR393, AT-N135Q-ΔS394 produces a significant decrease in anti-Xa activity of a pentasaccharide contained in plasma sample.

To estimate whether mutated antithrombin can be used as reversal of pentasaccharide in case of over-dosing, AT-N135Q-R393H and AT-N135Q-Pro394 are tested in Rotachrom® heparin assay for their ability to restore factor Xa activity. Under conditions of treatment for prevention of venous thromboembolic events, optimal pentasaccharide concentration ranges between 1.2 and 1.26 μg/mL in plasma. To mimic an over-dosing, pentasaccharide is added to human plasma pool at concentration of 3 μg/ml. Rotachrom® heparin assay is then performed by incubation of plasma over-dosed with pentasaccharide in the presence of mutated antithrombin in a ratio 1/1, 2/1, and 4/1 compared to plasma antithrombin concentration (i.e. 0.15, 0.3, 0.6 g/l). In this ex vivo assay, anti-factor Xa activity is proportional to the amount of pentasaccharide bound natural antithrombin present in tested plasma, which amount is directly proportional to pentasaccharide concentration in plasma. Therefore, effect of mutated antithrombin is expressed as a virtual decrease in pentasaccharide concentration that corresponds indeed to decrease in pentasaccharide bound natural antithrombin concentration (FIG. 7). AT-N135Q-R393H or AT-N135Q-Pro394 at concentration equal to plasmatic antithrombin concentration is able to significantly decrease anti-factor Xa activity to a level comparable to curative pentasaccharide treatment (from 3 μg/mL to virtually 1.04 μg or 1.16 μg/mL, respectively). When AT-N135Q-R393H is tested in 2 or 4 fold molar excess compared to natural antithrombin, virtual pentasaccharide concentration decreases to 0.49 or 0.29 μg/mL respectively (0.59 or 0.36 respectively with AT-N135Q-Pro394).

To estimate whether monomutated antithrombin can be used as reversal of pentasaccharide in case of over-dosing, AT-Pro394 is tested in Rotachrom® heparin assay for its ability to restore factor Xa activity. AT-Pro394 treatment is performed under the same conditions as for AT-N135Q-R393H and AT-N135Q-Pro394 when mimicking a pentasaccharide over-dosing. AT-Pro394 at concentration equal to plasmatic antithrombin concentration is able to significantly decrease anti-factor Xa activity. When AT-Pro394 is tested in 2 or 4 fold molar excess compared to natural antithrombin, virtual pentasaccharide concentration decreases to a level comparable to curative pentasaccharide treatment.

To evaluate the role of blood-vessel endothelium, or more precisely, the role of glycosamino-glycans exposed on endothelial cell surface, on competition between natural antithrombin and mutated antithrombin for pentasaccharide binding, the same experiment as described above is done but with pre-incubation of pentasaccharide containing plasma-mutated antithrombin mixture on HUVEC surface (FIG. 8). Pre-incubation on cell surface slightly influences effect of mutated antithrombin as reversal for pentasaccharide in case of over-dosing since in a ratio 1/1, 2/1, and 4/1 compared to plasma antithrombin concentration, AT-N135Q-R393H decreases anti-factor Xa activity from 3 μg/mL of pentasaccharide to virtually 1.66, 0.91 and 0.26 μg/mL respectively (compared to 1.04, 0.49 and 0.29 μg/mL in the absence of endothelial cell during pre-incubation). In the same conditions AT-N135Q-Pro394 decreases anti-factor Xa activity from 3 μg/mL of pentasaccharide to virtually 1.69, 0.94 and 0.27 μg/mL, respectively (compared to 1.16, 0.59 and 0.36 μg/mL in the absence of endothelial cell during pre-incubation).

Similarly, the same experiment as described above to evaluate the role of blood-vessel endothelium on competition between natural antithrombin and monomutated antithrombin for pentasaccharide binding is done, with pre-incubation of pentasaccharide containing plasma-monomutated antithrombin mixture on HUVEC surface. In the same conditions, pre-incubation on cell surface slightly influences effect of monomutated antithrombin as reversal for pentasaccharide in case of over-dosing, and AT-Pro394 decreases anti-factor Xa activity of pentasaccharide.

This ex-vivo competition experiment, strongly suggests that monomutated and mutated antithrombin can be used as reversal for pentasaccharide at a dose equal to plasma antithrombin in case of moderate over-dosing. A four molar excess of mutated antithrombin decreases about 10 times (or more) initial pentasaccharide concentration and is still compatible with therapeutic use in case of severe over-dosing.

c) Example of Anti Factor Xa Inhibitory Activity of the Mutated Antithrombins in Mouse Plasma

During the animal experimentation (see detailed protocol below), human mutated antithrombins compete with murine antithrombin for their binding to the pentasaccharide. Thus, preliminary in vitro assays are performed to determine the human mutated antithrombin concentration necessary to compete with the murine antithrombin present in mouse plasma supplemented with Fondaparinux.

These experiments are performed as described in section II-b, using mouse plasma instead of human plasma.

It is demonstrated that the mutated antithrombins compete with plasma antithrombin for pentasaccharide binding and are able to decrease significantly the plasma anti-Xa activity.

In the condition of the assay, addition of AT-R393H, AT-Pro394, AT-ΔR393-S394, AT-ΔR393, AT-ΔS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135 Q-ΔR393-S394, AT-N135Q-ΔR393, AT-N135Q-ΔS394 produces a significant decrease in anti-Xa activity of a pentasaccharide contained in mouse plasma.

It is demonstrated that mutated antithrombins compete with plasma antithrombin for pentasaccharide binding in human plasma and in mouse plasma. The amount of human mutated antithrombins required to compete with mouse antithrombins is equal or more important.

III/ In Vivo Characterization of the Mutated Antithrombins

The efficiency of the mutated antithrombins AT-R393H, AT-Pro394, AT-ΔR393-S394, AT-ΔR393, AT-ΔS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135Q-ΔR393-S394, AT-N135Q-ΔR393, AT-N135Q-ΔS394 as antidotes is evaluated in a murine model.

a) Animals

Animals (mice CF, males adults; or swiss mice, female adults (JANVIER)) are anesthetized using a protocol that does not modify the coagulation parameters, and in accordance with the European guidelines for animal experimentation.

The different drugs to be tested (fondaparinux, low molecular weight heparin, full length heparin and the mutated antithrombins) are administrated by intra-venous injection in the caudal vein to avoid the variability encountered when administration is performed subcutaneously. However, Heparin derivatives (fondaparinux, low molecular weight heparin and full length heparin) could also be administrated subcutaneously.

The blood is collected by punction in cave vein into tubes containing 0.105 mol/L trisodium citrate (1:10). Platelet-poor plasma is obtained by centrifugation at 2300 g for 10 minutes at 12° C. and stored at −80° C. until use.

b) Experimental Protocol

The following elements are determined:

-   -   the doses of fondaparinux inducing an overdose in mice,     -   the mutated antithrombins time course, and in particular the         time required to observe a maximal biological effect,     -   the minimal doses of mutated antithrombins necessary to         neutralize fondaparinux.

For each of these experiments, 4 groups of 5 mice each are used:

-   -   In the first group mice receive fondaparinux vehicle and then         antidote (mutated antithrombin) vehicle.     -   In the second group, mice receive fondaparinux and then antidote         vehicle     -   In the third group, mice receive fondaparinux vehicle and then         antidote     -   In the last group, mice receive fondaparinux and then antidote

1—Determination of the Doses of Fondaparinux Inducing an Overdose in Mice

Published data concerning fondaparinux pharmacokinetics in rats (Herbert J 4197), or experimental determination of fondaparinux pharmacokinetics in mice are used to develop the model.

In rat, fondaparinux has a maximal antithrombotic activity at doses of 100 nmol/kg (or 0.17 mg/kg).

Using this dose, the Cmax is 1.2±0.8 nmol/ml (or 2.1±1.4 mg/L). To induce an overdose, a 3 fold doses (300 nmol/kg, or 0.52 mg/kg) is tested. At this dose, the fondaparinux plasma concentration is about 3.5 to 4 nmol/ml (or 6 to 7 mg/L). An anti-Xa activity is determined to ensure that an overdose is obtained.

Anti FXa activity measurements is performed as previously described using a Stachrom heparin® kit on a STA (Stago, France) or a Rotachrom heparin® kit with procedure adapted for measurement in microplate reader (Dynatech MR 5000). A calibration curve with pentasaccharide is performed, allowing the expression of the results in nmol/ml (or in mg/L) of plasma.

2—Study of the Mutated Antithrombins Time of Action

In rats, fondaparinux has a Tmax of 5 min after intra-venous injection of 100 nmol/kg (or 0.17 mg/kg), or 30 min after subcutaneous injection of 100 nmol/kg (or 0.17 mg/kg).

In mouse, an overdose of fondaparinux is observed at 300 nmol/kg (or 0.52 mg/kg) but maximal concentration in plasma is observed less than 15 min after sub-cutaneous injection.

Thus, the antidote (mutated antithrombins) is administered intravenously, 5 min after the intra-venous injection of the fondaparinux, and 5, 15 or 30 min after the subcutaneous injection of the fondaparinux.

The mutated antithrombin time course is studied by measuring the plasma anti-Xa activity 5 min, 10 min, 20 min and 40 min after the antidote administration.

These experiments allow to determine the time of action of the antidote.

3—Determination of the Minimal Doses of Mutated Antithrombins Necessary for Fondaparinux Neutralization

Once the time of action determined, a dose response curve is performed in vivo to determine the dose of antidote necessary to obtain a decrease in Fondaparinux plasma concentration within the therapeutic range around 1.2 nmol/ml (or 2 mg/L), or around 0.7 nmol/ml (or 1.2 mg/L).

Using these experimental conditions, injection of mutated antithrombins (AT-R393H, AT-Pro394, AT-ΔR393-S394, AT-ΔR393, AT-ΔS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135Q-ΔR393-S394, AT-N135Q-ΔR393 or AT-N135Q-ΔS394), result in a significant decrease of the plasma anti FXa activity in mice treated with pentasaccharide. 

1-41. (canceled)
 42. Method for the prevention or treatment of pathologies linked to or associated with coagulation disorders with a drug involving a mutated antithrombin having substantially no activity.
 43. Method according to claim 42, wherein said mutated antithrombin has substantially no anticoagulant activity.
 44. Method according to claim 42, for the treatment or prevention of hemorrhagic disorders and related pathologies, resulting from side effects of said anticoagulant in association with an anticoagulant.
 45. Method according to claim 42, wherein said mutated antithrombin has the ability to bind to the anticoagulant and to shift, including in vivo, the binding between plasma antithrombin and said anticoagulant.
 46. Method according to claim 44, wherein said mutated antithrombin is in association with a mutant of antithrombin, the amino acid sequence of which differs from that of said mutated antithrombin, wherein said mutated antithrombin has the ability to shift, including in vivo, the binding between plasma antithrombin and said anticoagulant and the ability to shift, including in vivo, the binding between said mutant of antithrombin and said anticoagulant.
 47. Method according to claim 42, wherein said mutated antithrombin has substantially lost factor Xa inhibitory activity and thrombin inhibitory activity.
 48. Method according to claim 44, wherein the value of the dissociation equilibrium constant (Kd) of the complex resulting from the binding of said mutated antithrombin with said anticoagulant, at a given ionic strength, including at physiological ionic strength, is similar or lower than the value of the Kd of the complex between plasma antithrombin and said anticoagulant.
 49. Method according to claim 46, wherein the value of the Kd of the complex resulting from the binding of said mutated antithrombin with said anticoagulant, at a given ionic strength, including at physiological ionic strength, is similar or lower than the value of the Kd of the complex between said mutant of antithrombin and said anticoagulant.
 50. Method according to claim 44, wherein the anticoagulant is chosen in the group consisting of: heparins, heparins derivatives, including unfractioned heparins, low molecular weight heparins, the anticoagulant pentasaccharide (Fondaparinux), and its derivatives (Idraparinux: SANORG 34006), and heparinoids (Danaparoid sodium ORG 10172).
 51. Method according to claim 44, wherein the pathologies linked to or associated with coagulation disorders are arterial or venous thrombotic disorders consisting in pulmonary embolism, deep vein thrombosis, myocardial infraction, unstable angina, stroke, disseminated intravascular coagulation, or hemorrhagic disorders consisting in FVIII deficiency (hemophilia A), FIX deficiency (hemophilia B), FVII deficiency, FX deficiency, FXI deficiency, FII deficiency, vWF deficiency, acquired antibodies against these coagulation factors, fibrinolysis abnormalities, platelets abnormalities, disseminated intravascular coagulation and any pathology associated with a combination of these deficiencies or abnormalities.
 52. Method according to claim 44, wherein said mutated antithrombin comprises at least one mutation within the region from the amino acid at position 380 to the amino acid at position 400, including the region from the amino acid at position 380 to the amino acid at position 397, the amino acid numbering referring to the antithrombin amino acid sequence represented by SEQ ID NO: 2, said mutation being a substitution, insertion or deletion.
 53. Method according to claim 52, wherein said mutated antithrombin comprises at least one mutation within the region from the amino acid at position 390 to the amino acid at position 394, including position
 393. 54. Method according to claim 52, wherein said mutated antithrombin comprises a mutation at the amino acid at position
 393. 55. Method according to claim 52, wherein said mutated antithrombin further comprises at least one mutation at the glycosylation sites at the amino acid at position 96, 135, 155 or
 192. 56. Method according to claim 52, wherein said mutated antithrombin further comprises a mutation at the glycosylation sites at the amino acid at position
 135. 57. Method according to claim 52, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of: SEQ ID NO:4, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the substitution of the amino acid at position 393, by an Histidine (His), or SEQ ID NO:6, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the insertion of a Proline (Pro) between the amino acid at position 393 and the amino acid at position 394, or SEQ ID NO:8, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, or SEQ ID NO:10, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position
 394. 58. Method according to claim 52, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of: SEQ ID NO:14, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the substitution of the amino acid at position 393, by an Histidine (His), and the substitution of the amino acid at position 135, by a Glutamine (Gln), or SEQ ID NO:16, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the insertion of a Proline (Pro) between the amino acid at position 393 and the amino acid at position 394, and the substitution of the amino acid at position 135, by a Glutamine (Gln), or SEQ ID NO:18, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393 and at position 394, or SEQ ID NO:20, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393 and the substitution of the amino acid at position 135, by a Glutamine (Gln), or SEQ ID NO:22, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 394 and the substitution of the amino acid at position 135, by a Glutamine (Gln), or SEQ ID NO:24, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393 and at position 394, and the substitution of the amino acid at position 135, by a Glutamine (Gln).
 59. Method according to claim 42, wherein said mutated antithrombin comprises at least one mutation within the region from the amino acid at position 412 to the amino acid at position 432, the amino acid numbering referring to the antithrombin amino acid sequence comprising the signal peptide, represented by SEQ ID NO: 26, said mutation being a substitution, insertion or deletion.
 60. Method according to claim 59, wherein said mutated antithrombin comprises at least one mutation within the region from the amino acid at position 422 to the amino acid at position
 426. 61. Method according to claim 59, wherein said mutated antithrombin comprises a mutation at the amino acid at position
 425. 62. Method according to claim 59, wherein said mutated antithrombin further comprises at least one mutation at the glycosylation sites at the amino acid at position 128, 167, 187 or
 224. 63. Method according to claim 59, wherein said mutated antithrombin further comprises a mutation at the glycosylation sites at the amino acid at position
 167. 64. Method according to claim 59, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of: SEQ ID NO:28, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the substitution of the amino acid at position 425, by an Histidine (His), or SEQ ID NO:30, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the insertion of a Proline (Pro) between the amino acid at position 425 and the amino acid at position 426, or SEQ ID NO:32, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, or SEQ ID NO:34, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position
 426. 65. Method according to claim 59, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of: SEQ ID NO:38, said amino acid sequence comprising in the sequence of antithrombin represented by SEQ ID NO:26, the substitution of the amino acid at position 425, by an Histidine (His), and the substitution of the amino acid at position 167, by a Glutamine (Gln), or SEQ ID NO:40, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the insertion of a Proline (Pro) between the amino acid at position 425 and the amino acid at position 426, and the substitution of the amino acid at position 167, by a Glutamine (Gln), or SEQ ID NO:42, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425 and at position 426, or SEQ ID NO:44, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425 and the substitution of the amino acid at position 167, by a Glutamine (Gln), or SEQ ID NO:46, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 426 and the substitution of the amino acid at position 167, by a Glutamine (Gln), or SEQ ID NO:48, said amino acid sequence comprising, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425 and at position 426 and the substitution of the amino acid at position 167, by a Glutamine (Gln).
 66. Product comprising at least one mutated antithrombin having substantially no activity, including no anticoagulant activity, and at least one compound, including anticoagulant, as a combination product for a separate or sequential use in the prevention or treatment of hemorrhagic disorders and related pathologies resulting from side effects of said anticoagulant.
 67. Product according to claim 66, wherein said mutated antithrombin has the ability to bind to the anticoagulant and to shift, including in vivo, the binding between plasma antithrombin and said anticoagulant.
 68. Product according to claim 66, wherein said mutated antithrombin has the ability to shift, including in vivo, the binding between plasma antithrombin and said anticoagulant and the ability to shift, including in vivo, the binding between said mutant of antithrombin and said anticoagulant.
 69. Product according to claim 66, wherein said mutated antithrombin has substantially lost factor Xa inhibitory activity and thrombin inhibitory activity.
 70. Product according to claim 66, wherein the value of the dissociation equilibrium constant (Kd) of the complex resulting from the binding of said mutated antithrombin with said anticoagulant, at a given ionic strength, including at physiological ionic strength, is similar or lower than the value of the Kd of the complex between plasma antithrombin and said anticoagulant.
 71. Product according to claim 68, wherein the value of the Kd of the complex resulting from the binding of said mutated antithrombin with said anticoagulant, at a given ionic strength, including at physiological ionic strength, is similar or lower than the value of the Kd of the complex between said mutant of antithrombin and said anticoagulant.
 72. Product according to claim 66, wherein the anticoagulant is chosen in the group consisting of: heparins, heparins derivatives, including unfractioned heparins, low molecular weight heparins, the anticoagulant pentasaccharide (Fondaparinux), and its derivatives (Idraparinux: SANORG 34006), and heparinoids (Danaparoid sodium ORG 10172).
 73. Product according to claim 66, wherein the coagulation disorders are arterial or venous thrombotic disorders consisting in pulmonary embolism, deep vein thrombosis, myocardial infraction, unstable angina, stroke, disseminated intravascular coagulation or hemorrhagic disorders consisting in FVIII deficiency (hemophilia A), FIX deficiency (hemophilia B), FVII deficiency, FX deficiency, FXI deficiency, FII deficiency, vWF deficiency, acquired antibodies against these coagulation factors, fibrinolysis abnormalities, platelets abnormalities, disseminated intravascular coagulation and any pathology associated with a combination of these deficiencies or abnormalities.
 74. Product according to claim 66, wherein said mutated antithrombin is an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.
 75. Product according to claim 66, wherein said mutated antithrombin is chosen from the group consisting of: SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:48.
 76. Mutated antithrombin, chosen from the group consisting in mutated antithrombin which contains at least an amino acid insertion between the amino acid at position 393 and the amino acid at position 394, in the sequence of antithrombin represented by SEQ ID NO:2, including an insertion of a Proline (Pro), mutated antithrombin, which contains at least an amino acid insertion between the amino acid at position 425 and the amino acid at position 426, in the sequence of antithrombin represented by SEQ ID NO:26, including an insertion of a Proline (Pro), mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 393, in the sequence of antithrombin represented by SEQ ID NO:2, mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 425, in the sequence of antithrombin represented by SEQ ID NO:26, mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 394, in the sequence of antithrombin represented by SEQ ID NO:2, or mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 426, in the sequence of antithrombin represented by SEQ ID NO:26.
 77. Mutated antithrombin according to claim 76, wherein said mutated antithrombin is represented by a sequence chosen from the group consisting in: SEQ ID NO: 6, SEQ ID NO: 30, SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 10, or SEQ ID NO:
 34. 78. Mutated antithrombin, which contains at least two mutations: the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the substitution of the amino acid at position 393, by an Histidine (His), the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 14, the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the insertion of a Proline (Pro) between the amino acid at position 393 and the amino acid at position 394, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 16, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the deletion of the amino acid at position 394, said mutated antithrombin being in particular represented by SEQ ID NO: 18, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 20, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 394, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 22, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the deletion of the amino acid at position 394, and the third mutation being substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 24, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the substitution of the amino acid at position 425, by an Histidine (His), the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 38, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the insertion of a Proline (Pro) between the amino acid at position 425 and the amino acid at position 426, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 40, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the deletion of the amino acid at position 426, said mutated antithrombin being in particular represented by SEQ ID NO: 42, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 44, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 426, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 46, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the deletion of the amino acid at position 426, and the third mutation being substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO:
 48. 79. Nucleotide sequence encoding a mutated antithrombin, said mutated antithrombin being chosen in the group consisting in: mutated antithrombin which contains at least an amino acid insertion between the amino acid at position 393 and the amino acid at position 394, in the sequence of antithrombin represented by SEQ ID NO:2, including an insertion of a Proline (Pro), mutated antithrombin, which contains at least an amino acid insertion between the amino acid at position 425 and the amino acid at position 426, in the sequence of antithrombin represented by SEQ ID NO:26, including an insertion of a Proline (Pro), mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 393, in the sequence of antithrombin represented by SEQ ID NO:2, mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 425, in the sequence of antithrombin represented by SEQ ID NO:26, mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 394, in the sequence of antithrombin represented by SEQ ID NO:2, or mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 426, in the sequence of antithrombin represented by SEQ ID NO:26, or mutated antithrombin, which contains at least two mutations selected in the group consisting in: the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the substitution of the amino acid at position 393, by an Histidine (His), the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 14, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the insertion of a Proline (Pro) between the amino acid at position 393 and the amino acid at position 394, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 16, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the deletion of the amino acid at position 394, said mutated antithrombin being in particular represented by SEQ ID NO: 18, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 20, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 394, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 22, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the deletion of the amino acid at position 394, and the third mutation being substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 24, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the substitution of the amino acid at position 425, by an Histidine (His), the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 38, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the insertion of a Proline (Pro) between the amino acid at position 425 and the amino acid at position 426, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 40, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the deletion of the amino acid at position 426, said mutated antithrombin being in particular represented by SEQ ID NO: 42, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 44, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 426, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 46, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the deletion of the amino acid at position 426, and the third mutation being substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO:
 48. 80. Nucleotide sequence encoding a mutated antithrombin as defined in claim 79, wherein said nucleotide sequences are chosen in the group consisting of SEQ ID NO: 5, 7, 9, 13, 15, 17, 19, 21, 23, 29, 31, 33, 37, 39, 41, 43,
 47. 81. Pharmaceutical composition comprising as active ingredient a mutated antithrombin, wherein said mutated antithrombin is chosen in the group consisting in: mutated antithrombin which contains at least an amino acid insertion between the amino acid at position 393 and the amino acid at position 394, in the sequence of antithrombin represented by SEQ ID NO:2, including an insertion of a Proline (Pro), mutated antithrombin, which contains at least an amino acid insertion between the amino acid at position 425 and the amino acid at position 426, in the sequence of antithrombin represented by SEQ ID NO:26, including an insertion of a Proline (Pro), mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 393, in the sequence of antithrombin represented by SEQ ID NO:2, mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 425, in the sequence of antithrombin represented by SEQ ID NO:26, mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 394, in the sequence of antithrombin represented by SEQ ID NO:2, or mutated antithrombin, which contains at least an amino acid deletion of the amino acid at position 426, in the sequence of antithrombin represented by SEQ ID NO:26, or mutated antithrombin, which contains at least two mutations selected in the group consisting in: the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the substitution of the amino acid at position 393, by an Histidine (His), the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 14, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the insertion of a Proline (Pro) between the amino acid at position 393 and the amino acid at position 394, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 16, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the deletion of the amino acid at position 394, said mutated antithrombin being in particular represented by SEQ ID NO: 18, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 20, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 394, the second mutation being the substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 22, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:2, the deletion of the amino acid at position 393, the second mutation being the deletion of the amino acid at position 394, and the third mutation being substitution of the amino acid at position 135, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 24, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the substitution of the amino acid at position 425, by an Histidine (His), the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 38, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the insertion of a Proline (Pro) between the amino acid at position 425 and the amino acid at position 426, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 40, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the deletion of the amino acid at position 426, said mutated antithrombin being in particular represented by SEQ ID NO: 42, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 44, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 426, the second mutation being the substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO: 46, or the first mutation being, in the sequence of antithrombin represented by SEQ ID NO:26, the deletion of the amino acid at position 425, the second mutation being the deletion of the amino acid at position 426, and the third mutation being substitution of the amino acid at position 167, by a Glutamine (Gln), said mutated antithrombin being in particular represented by SEQ ID NO:
 48. in combination with a pharmaceutical acceptable vehicle.
 82. Pharmaceutical composition comprising as active ingredient a mutated antithrombin of SEQ ID NO: 6, 8, 10, 14, 16, 18, 21, 22, 24, 30, 32, 34, 38, 40, 42, 44, 46, 48, in combination with a pharmaceutical acceptable vehicle.
 83. Pharmaceutical composition, comprising as active ingredient a mutated antithrombin, of SEQ ID NO:4, 12, 28 or 36 in combination with a pharmaceutical acceptable vehicle. 